The release of preloaded radiolabeled norepinephrine ([3H]NE) from slices of rat hippocampus can be stimulated by excitatory amino acids that interact with the N-methyl-D-aspartate (NMDA) receptor. The acidic dipeptide N-acetyl-L-aspartylglutamate (NAAG) is colocalized with NE in the cell bodies of locus coeruleus (the origin of the noradrenergic projections to the hippocampus) and the hippocampus itself. The function of NAAG in these neurons has not been demonstrated, although evidence exists that it may serve as a neuromodulator in other neuronal pathways. NAAG inhibited the release of [3H]NE stimulated by NMDA and L-glutamate in a concentration-related manner. The maximal inhibition produced by NAAG was about 25% of the control release stimulated by 25 microM NMDA. The effects observed were caused by the intact dipeptide and not the degradation artifacts produced by the enzyme N-acetylated-alpha-linked-acidic dipeptidase because N-acetyl-L-aspartate had no significant effect on the release and L-glutamate was stimulatory. The activity of this enzyme appears to be suppressed under the assay conditions used. Although the addition of glycine did not enhance NMDA-stimulated release, 7-chlorokynurenate and 1-hydroxy-3-amino-pyrrolidone-2 decreased the release in a concentration-dependent manner. Furthermore, the attenuation produced by NAAG plus 7-chlorokynurenate or 1-hydroxy-3-aminopyrrolidone-2 was greater than the inhibitory actions of either glycine antagonist alone. Similarly, NAAG produced additional inhibition over that produced by either of two different voltage-dependent calcium channel blockers.(ABSTRACT TRUNCATED AT 250 WORDS)